HIV-1 subtype diversity and immuno-virological outcomes among adolescents failing antiretroviral therapy in Cameroon: A cohort study

Objective We sought to evaluate the variability of HIV-1 and its effect on immuno-virological response among adolescents living with perinatally acquired HIV (APHI). Methods A cohort study was conducted from 2018–2020 among 311 APHI receiving antiretroviral therapy (ART) in Cameroon. Sequencing of protease and reverse transcriptase regions was performed for participants experiencing virological failure, VF, (Plasma viral load, PVL ≥ 1000 RNA copies/ml). HIV-1 subtypes were inferred by phylogeny; immuno-virological responses were monitored at 3-time points (T1-T3). Cox regression modeling was used to estimate adjusted hazard ratios (aHRs) of progression to: CD4 < 250, and PVL > 5log10, adjusted for acquired drug resistance, gender, ART line, adherence, and duration on treatment; p < 0.05 was considered statistically significant. Results Of the 141 participants in VF enrolled, the male-female ratio was 1:1; mean age was 15 (±3) years; and median [IQR] duration on ART was 51 [46–60] months. In all phases, 17 viral clades were found with a predominant CRF02_AG (58.2%, 59.4%, and 58.3%). From T1-T3 respectively, there was an increasing CD4 count (213 [154–313], 366 [309–469], and 438 [364–569] cells/mm3) and decline log10 PVL (5.23, 4.43, and 4.43), similar across subtypes. Among participants with CRF02_AG infection, duration of treatment was significantly associated with both rates of progression to CD4 < 250, and PVL > 5log10, aHR = 0.02 (0.001–0.52), and aHR = 0.05 (0.01–0.47) respectively. Moreover, four potential new HIV-1 recombinants were identified (CRF02_AG/02D, CRF02_AG/02A1F2, D/CRF02_AG, and AF2/CRF02_AG), indicating a wide viral diversity. Conclusion Among APHI in settings like Cameroon, there is a wide genetic diversity of HIV-1, driven by CRF02_AG and with potential novel clades due to ongoing recombination events. Duration of treatment significantly reduces the risk of disease progression.


Introduction
HIV molecular surveillance reveals recombinant forms account for almost a quarter of HIV infection worldwide [1].West-Central Africa is believed to be the geographical origin of HIV-1, with the Congo basin being the most likely birthplace of HIV-1 groups M, N, O, and P [2,3].This is perhaps the reason why it has one of the most genetically diverse HIV epidemics on the globe.Moreover, the circulation of virtually every known HIV-1 group M pure subtype, many circulating recombinant forms (CRFs), and a variety of unique recombinant forms (URFs) has been observed within this geographical region [4].The most widely circulating variant in this region is the CRF02_AG viral clade.
There is increasing evidence that the extreme variability and high evolution rate of HIV-1 favors the emergence of HIV antiretroviral drug resistance (HIVDR).In addition, some recombinant forms, such as the CRF01_AE variant in China, have been shown to have a faster rate of disease progression, and better replicative fitness when compared to pure lineages [5].In a context of wide genetic diversity among adolescents living with perinatally acquired HIV (APHI) [6], it is therefore essential to monitor and genetically characterize HIV on a global scale.Special attention has to be paid to hot spot regions with wide HIV genetic variability like Cameroon, noting that subtype variability and the extent of viral diversity within a geographical region considerably impact HIV diagnosis, treatment outcome, and hence prevention and management [4,7,8].
The increase in HIV-1 viral diversity because of the relative adaptation of the virus to a given host environment constitutes a factor that is particularly relevant to the emergence of drug-resistant variants.This provides a unique opportunity to study and monitor the evolution of HIV-1 strains and the clinical implications of these on the treatment, and immune/ virological response among adolescents [9][10][11].
The determination of HIV subtypes is based on comparing a query sequence to a set of reference sequences and obtaining the best match as the putative subtype.A wide array of currently used subtyping tools rely on an initial alignment step to measure similarity with the reference set.The population of APHI is of particular interest because of the long time after infection (starting from an established time point, that is the birth), and as such viral adaptation to the host is likely to occur.
In this study, we evaluated the effect of HIV-1 genetic diversity on immune-virological response and described potential emerging resistant variants among APHI failing first-and second-line antiretroviral treatment.Specifically, we sought to assess HIV-1 genetic variability among these APHI, evaluate the immune-virological response according to HIV-1 subtypes, determine the trends of virological response according to HIV-1 subtypes, and describe the selection of potential resistant variants among these adolescents.

Study design
A cohort study was conducted from 2018-2020 among 311 adolescents living with perinatally acquired HIV (APHI) receiving antiretroviral therapy (ART) in one of the selected health facilities within the "Resistance Evolution among Adolescents in Yaoundé and its surroundings" (READY-study) in the Centre region of Cameroon.Participants were recruited following consecutive, and exhaustive sampling.Follow-up was performed at enrollment (T1), at 6 months (T2), and at 12 months (T3).Included in the current analysis were only participants failing first-and second-line ART with plasma viral load (PVL) � 1000 RNA copies/ml (Table 1).

Inclusion and exclusion criteria
Included in the study were APHI; aged 10-19 years, receiving a standard reverse transcriptase inhibitor-based first-or ritonavir-boosted protease inhibitor-based second-line ART regimen for at least 6 months, provided written assent, and informed consent from their legal guardian (s), and irrespective of exposure to the prevention of mother to child transmission (PMTCT) antiretrovirals.APHI who were not formally registered in the ART monitoring system of any study site, reported to be ART-naïve, or on a drug regimen not included in the national guidelines, or structured treatment interruption were not enrolled.Moreover, participants who freely withdrew from the study, or were transferred out of a study site before mid-or endpoint were excluded.

Sample size
The minimal sample size was established following statistical calculations [12].Assuming the rate of VF at 40%, a 95% confidence interval, and 80% statistical power, the sample size was 174 participants.Adding 10% potential loss to follow-up (LTFU) during the one-year study period and 20% sequencing failure rate, the minimal sample size was 243 APHI, majoring to 250 total sample size, further stratified into 150 APHI in the referral centers (75 per site), and 100 in the rural HIV management unit settings (50 per site), as per coverage in ART in these two geographical locations.Effectively 311 participants were enrolled, 213 from referral centers and 98 from management units.Of these, the current analysis included 141 participants (with PVL � 1000 RNA copies/mL) who had sequence data available from enrolment to 12-month follow-up.
The major outcomes from the study were the viral genetic diversity among APHI, immunevirological responses stratified by subtype, the trends of virological response stratified by subtype, and the potential emerging resistant variants among these APHI failing treatment.Adequate immunological status was defined as absolute CD4 count � 250 cells/mm 3 and Immunological failure (IF) as < 250 CD4 cells/mm 3 [17].PVL was classified as high viral load (PVL > 5log 10 RNA copies/ml), and low viral load (PVL < 5 log 10 ) [18].Genotypic resistance testing (GRT) was performed for these participants failing treatment.

Data analysis
Data were analyzed using SPSS version 22, with p < 0.05 considered statistically significant.A comparison of means and medians of various characteristics was done using the paired t-test.Chi-square and Fisher's exact tests were used for determining associations between qualitative variables.Study endpoints were defined as CD4 count < 250 cells/mm 3 and PVL > 5 log RNA/mL.Cox proportional hazard regression models were used to estimate hazard ratios (HRs) of disease progression to each endpoint stratified by HIV-1 subtype, adjusting for the presence of drug resistance mutations, gender, ART line, adherence, and duration on ART.Kaplan Meier curves were used to examine time to CD4 count decline to < 250 cells/mm 3 and VL > 5 log 10 RNA copies/mL on ART across subtypes, with the use of the log-rank test to test the significance of observed differences between groups.Survival curves were drawn using SPSS version 22.Moreover, median CD4 cell rise and PVL decline over time were determined and presented as figures.

Ethical statement
Administrative authorizations were obtained from the Directors of the health facilities used in this study.Moreover, an ethical clearance was obtained from the Cameroonian National ethics committee for research on human subjects (clearance 2018/01/981/CE/CNERSH/SP) in Yaounde ´, Cameroon.Written informed consent and assent were obtained from the parents or legal guardian(s), and every participant respectively.Laboratory results of viremia, CD4 cell count, and GRT were freely delivered to each participant for their clinical benefits, data management was under strict confidentiality by using unique identifiers, and access to data was password protected.
At 6-months post-enrolment, improving values in median CD4 count were observed with a higher value among non-CRF02_AG clades (426 ), in comparison to the CRF02_AG variant (345 [186-600]).These were accompanied by a declining trend in median log 10 PVL, with a lower value observed in participants infected with the CRF02_AG variant (4.37 [3.86-5.13]),relative to those with non-CRF02_AG clades (4.48 [3.77-5.06]).Likewise, this difference was not statistically significant with respect to genetic variability (Table 3).The proportion of participants who had an adequate immunological response, as well as those with PVL < 5 log was comparable across subtypes with p-value > 0.05 (Tables 4 and 5).
Concerning median CD4 count, all subtypes showed a consecutive rise over time (Fig 2b).Likewise, a steady decrease in median PVL was observed from enrolment to the end of the observation period (Fig 2c).At the end of the study, no statistically significant differences with respect to subtype were observed in both participants who experienced adequate immunological response (CD4 count � 250 cells/mm 3 ), and those with PVL< 5 log 10 (Tables 4 and 5).
Over the entire study period, the duration of ART was significantly associated with a decrease in the probability of experiencing poorer immunologic outcomes among participants infected by the CRF02_AG variant at any given time over the two-year duration of the study [adjusted hazard ratio (aHR) = 0.02, 95% confidence interval (95% CI): 0.001-0.52,and p = 0.017].Similarly, there was a statistically significant association between the duration of treatment and the probability of a decline in PVL in participants with CRF02_AG infection [aHR = 0.05, 95% CI: 0.01-0.47,and p = 0.008] (Table 7).
Assessing potentially emerging HIV-1 genetic variants, or unique recombinant forms (URF) associated with reduced viral susceptibility to currently available ARV drugs in the Cameroonian context, using four rapid subtyping tools revealed a high rate of inter-subtype recombination, confirmed by phylogenetic analyses (Fig 3c ), with the identification of eight discordant potential unassigned URFs.From these, further analyses to detect and analyze recombination and/or genomic assortment signals using RDP4 revealed four potential URFs with distinct breakpoints, each associated with major drug resistance mutations mainly driven by the ARV drug classes of non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) (Table 8).Further phylogenetic analysis suggest the presence of inter-subtype recombinants; recombinant CRF02_AG/02D, recombinant CRF02_AG/02A1F2, recombinant D/CRF02_AG, and recombinant AF2/CRF02_AG (Fig 3d).

Discussion
Our study was conducted among adolescents living with perinatally acquired HIV (APHI) in urban and rural settings of the Center region of Cameroon.The predominant genetic variant was CRF02_AG, this is similar to other studies carried out in Cameroon [4,6,9,[19][20][21].The CRF02_AG variant is the most prevalent HIV-1 viral clade in West Africa [1].It is a recombinant of pure subtypes A and G, which are both associated with slower disease progression to AIDS [5].The higher infectivity observed in infection with this variant may be attributed to its enhanced replicative fitness over its pure subtype constituents and hence improved adaptation  to its host.HIV-1 subtypes C, D, and the genetic variant CRF01_AE observed in our study have been associated with faster HIV-1 disease progression to AIDS [7,10,22,23] and advanced immunodeficiency.Evidence suggests that recombinant forms have a faster rate of disease progression when compared to pure lineages.This is because they have better replicative fitness as compared to their parental forms [5].However, the immune-virological response in this evaluation was similar across viral clades, despite the presence of a wide array of circulating recombinant forms and unique recombinant forms (CRF18_cpx, CRF49_cpx, G/J, CRF A1/G, CRF G/A, CRF11_cpx, CRF13_cpx, CRF37_cpx).In this study, there was no statistically significant difference across subtypes with respect to CD4 cell count at enrolment.Likewise, there was no significant difference in the proportion of participants who achieved adequate immunological response at 12 months post-enrolment.These results were similar to those obtained in other studies [24,25].Moreover, assessing endpoint events in our study revealed that duration on ART was significantly associated with the rate of progression to poorer immunological outcomes among participants with CRF02_AG infection.It should be noted that the focus of the majority of studies of inference on the effect of subtype on immunologic decline has been focused on patients' pre-exposure to ART at baseline.Additionally, most focus on comparing patients harboring subtype B versus non-B subtypes, hence grouping minority variants into non-B subtypes.This makes it difficult to assess the effect of these less predominant viral clades.Studies carried out in China, where variant CRF01_AE is the most predominant clade, revealed that this variant is associated with fast progression to AIDS (with a shorter median time from the estimated date of seroconversion to AIDS) and advanced immunodeficiency (shorter median time from estimated date of seroconversion to CD4 cell count <100 cells/μl) as compared to non-CRF01_AE viral clades [22,23].However, a study in Nigeria reported similar rates of CD4 cell count recovery across all subtypes [24], which is concordant with the results obtained in our evaluation.In contrast, a previous study revealed that patients infected with either a CRF02_AG strain or another non-B viral clade had better immunological responses than those infected with a subtype-B virus [26].Despite the absence of statistical significance in the rate of progression to poorer immunologic outcome in this evaluation, HIV-1 genotypes such as subtypes C and D, and variant CRF01_AE were identified in our study population.These have previously been associated with faster disease progression [22,23,27], likely due to faster rates of CD4 T-cell decline.Additionally, a higher probability of having a virus with a CXCR4 tropism in subtype D infections has been reported.Conversely, subtypes A and G have been associated with a less aggressive disease progression [7].Further analysis into the rise in median CD4 cell count over time, as well as the estimated time to achieve favorable immunologic response in our study, showed similar responses across all subtypes.These results agree with those of a previous study carried out in a West African country [24].However, they are discordant with results obtained in geographical regions outside West-Central Africa [26][27][28][29].Virological responses at enrolment, 6-, and 12-month follow-ups on treatment were comparable across all subtypes.These were similar to results obtained by Ogbenna et al [24] but in disagreement with those obtained by Chaix et al in France where infection with CRF02_AG and other non-B viral clades conferred better virological responses than patients harboring Bsubtypes [26].These findings of similar virological responses across subtypes are consistent with those observed in previous studies that compared the responses to ART stratified by HIV-1 subtype [24,26,28,30].Our study also showed a continuous decrease in median PVL over time, as well as comparable overall virologic responses across subtypes.Similar observations have been reported in other studies [24,26,28,29].
In our study, we identified four sequences with discordant subtypes according to the rapid subtyping tools used, which we classified as potential URFs.In addition, each of these sequences was associated with at least one major drug-resistant mutation.These results are in agreement with those observed in several other studies carried out in Cameroon [4,9,19,31,32].This study provided insight into the evolving HIV-1 diversity, the genomic complexity of URF infections, the structural dynamics of emerging recombinant forms, and possible implications for antiretroviral treatment.Thus, underlining the necessity of performing full/nearfull length next-generation sequencing to achieve accurate HIV surveillance, subtyping, and genotypic analyses.
As generally observed in longitudinal studies, there were some missing laboratory data over time, as well as loss to follow-up of participants by the end of the study which made it difficult to follow-up the entire sample population.These may have had an impact on data quality and the overall impact of the study findings on the target population.Furthermore, limiting sequencing only to samples that had PVL � 1000 RNA copies/ml and sequencing failure of some samples led to an underestimation of HIV genotypes.Additionally, further analyses like Simplot++ ought to be performed to investigate sequence similarity and confirm the detection of recombination events.However, the multi-time point follow-up period, the personalized monitoring, and repeated genotyping of participants to ensure within-program comparison of subtypes in a bid to limit expected confounders are the strengths of this study.

Conclusion
This study revealed a wide variety of HIV-1 genotypes with CRF02_AG predominance.Among participants with CRF02_AG infection, duration on antiretroviral treatment was significantly associated to both rates of progression to poorer immunological outcomes, and higher PVL following treatment failure.Virological outcomes were similar across HIV-1 subtypes/variants.Therefore, provided adequate observance of treatment, and personalized follow-up of this underserved adolescent population with particular importance given to immunological follow-up, current ART regimens have similar efficacy irrespective of subtype.

Fig 1 .
Fig 1. a: Trends of HIVDR across time points.Blue line: the decreasing trend of HIVDR over time.Error bars denote standard error.b: Trends of HIV drug resistance with respect to antiretroviral drug classes.Error bars denote standard error.https://doi.org/10.1371/journal.pone.0293326.g001

Fig 2 .
Fig 2. a: Rich genetic diversity of HIV-1 subtypes and genetic variants in the study population.b: Median CD4 count rise over time across subtypes.Error bars denote standard error.c: Median log plasma viral load decreases over time.Error bars denote standard error.https://doi.org/10.1371/journal.pone.0293326.g002

Fig 3 .
Fig 3. a: Kaplan Meier analysis of time estimate to reach CD4 count < 250 cells/mm 3 .b: Probability of plasma viral load > 5 log RNA copies/mL over time.c: Maximum likelihood phylogenetic tree of the Protease-Reverse transcriptase gene regions of study participant sequences.This depicts broad genetic diversity with a wide inter/intra subtype recombination.d: Splits Tree analysis results: Maximum likelihood phylogenetic tree of the Protease-Reverse transcriptase gene regions of the four (CNPS029, CME125, HDMF009, and HDMB015) inter-subtype recombinants identified in the study.https://doi.org/10.1371/journal.pone.0293326.g003